Our long range goal is to gain a better understanding of the regulatory mechanisms in smooth muscle. Smooth muscle is essential for normal body function and is the contractile element of arteries, veins, intestines, uterus, etc. It is known that an increase in the intracellular concentration of CA++ induces contraction of smooth muscle. The mechanism by which the changes in CA++ concentration are detected by the contractile apparatus and then processed to result in either contraction or relaxation is still controversial. There are two basic theories: the most popular hypothesis is that phosphorylation of myosin by a myosin light chain kinase (MLCK) activates the contractile apparatus and leads to contraction, deactivation (relaxation) is achieved by dephosphorylation of myosin by a myosin light chain phophatase (MLCP); the alternative theory is that regulation is achieved by a mechanism termed leiotonin and does not involve myosin phosphorylation. There is considerable evidence to indicate that the phosphorylation theory forms at least part of the regulatory mechanism and the focus of this proposal is to examine in detail different aspects of this theory. The association of the MLCK with the contractile elements will be studied. Evidence suggests that MLCK binds to actin. This will be confirmed and the conditions of binding established. If the MLCK is restricted to thin filaments the extent of filament overlap will dictate the level of phosphorylation. Improved procedures for the isolation of MLCK will be investigated. There are some data to sugget that all previously isolated MLCKs may be derived from a larger precursor. The phosphorylation reaction will also be analyzed in more detail since it is possible that phosphorylation is sequential rather than random, and whether this is a general property of myosin molecules will be tested by studying phosphorylation of smooth muscle and skeletal muscle muscle myosins and their subfragments. An important objective is to estgablish the role of phosphorylation and several experiments are suggested in which phosphorylation can be correlated to ATPase activity of actomyosin or superprecipitation. Whether or not non-cycling myosin-actin attachments are formed under in vitro conditions will be investigated. The data from the above experiments will be evaluated to assess the role of myosin phosphorylation and to question whether additional mechanisms are indicated. The latter will also be tested using CA++ insensitive MLCK.